Diagnostic testing process and apparatus

ABSTRACT

A method and apparatus for use in a flow through assay process is disclosed. The method is characterised by a “pre-incubation step” in which the sample which is to be analysed, (typically for the presence of a particular protein), and a detection analyte (typically an antibody bound to colloidal gold or a fluorescent tag) which is known to bind to the particular protein may bind together for a desired period of time. This pre incubation step occurs before the mixture of sample and detection analyte come into contact with a capture analyte bound to a membrane. The provision of the pre-incubation step has the effect of both improving the sensitivity of the assay and reducing the volume of sample required for an assay. An apparatus for carrying out the method is disclosed defining a pre-incubation chamber for receiving the sample and detection analyte having a base defined by a membrane and a second membrane to which a capture analyte is bound. In one version the pre-incubation chamber is supported above the second membrane in one position but can be pushed into contact with the membrane carrying the capture analyte thus permitting fluid transfer from the incubation chamber through the capture membrane. In another version the membrane at the base of the incubation chamber is hydrophobic and its underside contacts the capture membrane and when a wetting agent is applied to the contents of the pre-incubation chamber fluid transfer occurs.

RELATED APPLICATIONS

This application is the U.S. National Phase of International ApplicationNo. PCT/AU02/01119, filed 20 Aug. 2002, published in English, whichapplication claims priority under 35 U.S.C. § 119 or 365 to AustralianApplication No. PR 7144, filed 20 Aug. 2001, Australian Application No.PR 9451, filed 12 Dec. 2001, and Australian Application No. 2002950212,filed 11 Jul. 2002. The entire teachings of the above applications areincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to a diagnostic testing process and in particularto an apparatus for use in carrying out an assay process and to a methodof carrying out an assay process using that apparatus.

BACKGROUND OF THE INVENTION

Lateral flow and flow-through technology have been used for diagnosticassays for almost twenty years. Lateral flow technology is currentlydominant because lateral flow devices are easy to produce and the assaycan be performed in a simple 2-step process that can be adapted forwhole blood separation. This results in a simple device that can be usedin the field as a rapid point-of-care diagnostic (Cole et al 1996Tuberc. Lung. Dis. 77:363–368). However, multiple disease diagnosisusing lateral flow technology is very difficult because of differencesin lateral diffusion between samples and variation in flow rates betweenbatches of the partitioning membrane. This means that antigen orantibody signal strengths may vary both within tests and between batchesof tests, resulting in inconsistent results.

Existing flow-through diagnostic tests can be completed in less than twominutes compared with typical times of five to fifteen minutes forlateral flow tests. This advantage in speed however, is often at theexpense of sensitivity. A further disadvantage is that higher volumes ofsample are required to achieve the same sensitivity as lateral flow.This may be problematic in some situations. For example, the diagnosisof analytes (reagents) in whole blood requires the separation of plasmafrom whole blood cells. The higher volumes of whole blood required forthis would quickly block the membranes in the flow-through format.

The basic principal of flow-through assays is well established. Thetests are designed to determine the existence of, and in some cases, thequantity of, a predetermined analyte/reagent in a sample. Often thereagent will be a protein but other reagents can be tested for. If theassay is to test for the existence of a particular disease in a patient,the patient's body fluids may be tested for an antibody or other proteinproduced by the patient in response to the infection, or for a proteinwhich is expressed by the bacterium or viral agent or the like causingthe disease. In a typical flow through assay a liquid sample which isbelieved to contain the reagent is sucked into an absorbent pad via amembrane to which is bound a capture analyte which is known to bind tothe reagent. The membrane is then typically washed with a buffer and aliquid containing a detection analyte which also binds to the reagentand which includes a tracer or marker which is detectable, is applied tothe membrane. The detection analyte binds to the immobilised reagentbound to the membrane and can be seen or otherwise detected to indicatethe presence of the reagent.

U.S. Pat. No. 4,246,339 discloses a test device for assaying liquidsamples for the presence of a predetermined reagent. The device includestelescoping top and bottom members defining a liquid reservoirtherebetween and resilient means for biasing the members in the openposition. The top member defines a series of test wells each of whichhas a base defined by a microporous membrane with a capture analyteimmobilised on the membrane surface. Absorbent means are located in thebottom member, spaced from the membrane in the open position but incontact therewith in the closed position. U.S. Pat. No. 4,246,339discloses the adding the test serum diluted with a buffer to a testwell, and incubating the device at room temperature for ten minutesprior to depressing the cassette to the closed position to pass thesample through the membranes into the absorbent material. When themembranes are dry, the membrane is washed and then covered with asolution containing a detection analyte which binds to the immobilisedreagent followed by a subsequent step in which a stain is applied.

It will be appreciated that the process described in U.S. Pat. No.4,246,339, is a somewhat long drawn out, time consuming and tediousprocess and also lacks sensitivity.

A more recent flow through device is described in U.S. Pat. No.5,185,127 which discloses an assay device including a filter stack andan enclosure having a base portion and a lid. The filter stack has ahydrophilic membrane having a capture analyte thereon, referred to inU.S. Pat. No. 5,185,127 as a binder. A hydrophobic membrane is locatedunder the hydrophilic membrane and a pad of absorbent material islocated under the hydrophobic membrane. The lid includes an upwardlyextending rib which defines a recess having an insert therein. In use, asample containing the reagent (referred to in U.S. Pat. No. 5,185,127 asthe analyte) is placed in the well of the assay device at which time thereagent/analyte binds to the capture analyte/binder. Flow of the assaysolution however, does not take place because the aqueous solution doesnot wet the hydrophobic membrane placed under the hydrophilic membranein the filter stack. Thus as much time is necessary to complete thebinding of the detection analyte to the reagent is allowed. When bindingis judged to be complete, flow may be initiated by adding a wettingagent which wets the hydrophobic membrane. After which time the aqueousliquid flows into pad of absorbent material. The membrane may then bewashed and treated with a detection analyte/tracer which may be anantibody which specifically binds to the analyte, the antibody having alabel covertly conjugated thereto. Again the sensitivity of U.S. Pat.No. 5,185,127 is lacking and is not equivalent to that obtainable inlateral flow or ELISA formats.

It is one object of the present invention to provide an improved methodand apparatus for use in an assay process such as an immunoassay,diagnostic assay or the like in which the process and apparatus arecapable of screening a wide range of samples such as whole blood,plasma/serum, or samples with a high particulate load such as crushedgrain (eg wheat heads) and which is simple and rapid to perform whilststill maintaining sensitivities at least equivalent to that obtainablein lateral flow or ELISA formats.

A related object of the present invention is to provide a method andapparatus which can be successfully used for multiple disease diagnosisfrom a single whole blood or other sample in a single test. An extensionwould be successful screening of a sample in a single test for thepresence of multiple analytes not necessarily related to disease (e.gdrugs, agriculture, veterinary testing).

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed in Australia orelsewhere before the priority date of each claim of this application.

Because the prior art is not consistent in its terminology, for theavoidance of doubt and for the purpose of clarity, the following termsused in the specification below, are defined as follows. The term“reagent” is used to refer to the compound protein or the like which isto be detected by the assay. The term “capture analyte” is used to referto a compound which is bound to a membrane and to which the reagent willbind. The term “detection analyte” is used to refer to a compound whichwill also bind to the reagent and which carries a tracer or some otherelement whose presence may be detected, typically visually detectedwhether under visible light, or fluorescence.

SUMMARY OF THE INVENTION

In a first broad aspect, the present invention provides an apparatus andmethod for use in an assay process which is characterised by providing a“pre-incubation step” in which the sample and detection analyte (whichmay typically be an antibody bound to colloidal gold or a fluorescenttag) may bind together, which has the effect of both improving thesensitivity of the assay and reducing the volume of sample required foran assay prior to reaction of the sample/analyte complex with a reactionmembrane to which one or more ligands are bound.

Thus, in one aspect of the present invention there is provided anapparatus for use in an assay process comprising:

a first member comprising a first, porous, membrane to which is bound acapture analyte for binding to a reagent to be detected, the memberhaving an upper surface and a lower surface;

a second member being a body of absorbent material disposed below andtouching the lower surface of the first member;

a vessel for containing a liquid sample spaced above the first membersaid vessel having side walls and a base, the base being defined by asecond membrane, the vessel being capable of retaining a liquid samplefor a predetermined incubation period; and

means for supporting the vessel above the first member in two positions,a first position in which the membrane is spaced a sufficient distancefrom the first member so as to not permit fluid transfer from the vesselto the body of absorbent material, and a second position in which thesecond membrane is in contact with the first member, such contactpermitting fluid transfer from the vessel through the first and secondmembranes to the body of absorbent material.

In a related aspect the present invention provides a method for assayingfor the presence of a pre-determined reagent in a liquid samplecomprising the steps of:

a) providing a first porous membrane to which capture analytes forbinding to the reagent have been bound;

b) placing a sample to be assayed and a detection analyte in a vesselhaving a base defined by a second porous membrane, the vessel beingcapable of retaining the liquid sample for a predetermined incubationperiod;

c) allowing a sufficient period of time to pass for the detectionanalyte to bind to the reagent, if present in the liquid sample;

d) contacting the base of the vessel with the first porous membrane; and

e) causing the liquid sample to flow through the membranes to allow thereagent to bind to the capture analyte carried on the first membrane.

Thus, the present invention provides a chamber which may serve as apre-incubation chamber in which a pre-incubation step can occur wherethe sample and detection analyte combine, which improves the sensitivityof the test and reduces the volume of sample required for the assay. Ithas been found that the pre-incubation step increases the testsensitivity for a typical existing flow-through apparatus byapproximately ten times to equivalent levels of sensitivity comparedwith lateral flow technology, while still allowing the assay to becompleted in around two minutes compared to 10 minutes for lateral flowformats.

For example a ground wheat head suspension may be solubilised, and mixedand pre-incubated in the chamber with a detection analyte in the form ofan antibody against alpha-amylase linked to a colloidal gold particle.The contents of the chamber may then be allowed to flow through to thefirst membrane containing a capture analyte in the form of animmobilised anti-amylase antibody, and anti-amylase antibody/goldcomplexes will bind to the immobilised antibody forming a detectablesignal. The signal can be detected by the removal of the pre-incubationunit and washing of the reaction membrane with buffer.

This format can also be used for detecting reagents in whole blood sincewhole red blood cells can be removed in the pre-incubation chamber andthe plasma allowed to flow-through to the reaction membrane containing abound capture analyte. In this format, the base membrane defined at thebase of the pre-incubation chamber will typically be a membrane whichhas the correct pore size to retain the red blood cells and allow theplasma to pass through on contact with the first membrane. Similarlyparticulate samples containing grain extracts, cell or microbialextracts can be analysed with this flow-through format since particulatematter can be removed in the pre-incubation chamber and therefore cannotblock the reaction area on the upper surface of the reaction membrane.

The apparatus can also be used for detecting analytes in body fluidsother than blood, such as plasma, sera, urine, saliva and sputum. Inthis case, the sample can be retained in the pre-incubation chamber byuse of a hydrophobic membrane. To obtain efficient flow throughcapillary action to the second member when the pre-incubation chamber islowered, the reaction membrane is pre-wet with a wetting agentcontaining a detergent or the reaction membrane is blocked with ahygroscopic solution such as sucrose, trehalose, fructose, oralternatively, glycerol.

This changes the characteristics of the reaction membrane from anon-hygroscopic to a hygroscopic membrane allowing the sample to flowthrough to the second member upon contact of the membrane at the base ofthe pre-incubation chamber with the reaction membrane.

In a yet further embodiment, if a hydrophobic membrane is used as thebase of the pre-incubation chamber, the apparatus may be used with thehydrophobic membrane and reaction membrane in contact, with the operatoradding a wetting agent to the sample to cause flowthrough, when desired.

Thus, in a related aspect, there is provided an apparatus for use in anassay process comprising a housing including:

a first member comprising a first, porous, membrane to which is bound acapture analyte for binding to a reagent to be detected, the memberhaving an upper surface and a lower surface;

a second member being a body of absorbent material such as tissue paperor the like disposed below and touching the lower surface of the firstmember;

a chamber located above the first member said chamber having side walls,and a base including a second, hydrophobic, membrane, having an upperand a lower surface, the pre-incubation chamber being supported abovethe first member with the lower surface of the hydrophobic membrane incontact with the upper surface of the first member.

The pre-incubation chamber can also be used to remove analytes that mayinterfere with the assay, such as human anti-mouse antibodies (HAMAS),in solution or by binding anti-analyte antibodies to the surface of thechamber. The chamber can also be used to extract the analyte of interestfrom an absorbent surface such as a swab, which has been taken from thethroat of a patient, by swirling the swab in an extraction solution inthe chamber. The pre-incubation chamber may be part of a pre-filter unitwhich acts also to pre-filter the sample prior to contact with the uppersurface of the first member.

Examples of assays that can be preformed by this method where tworeaction steps are involved (the incubation of the analyte with thelabeled anti-analyte followed by the binding of this complex to asolid-phase anti-analyte), are:

-   Direct antigen assay    -   1. Ag* (analyte)+Ab*₁ (anti-Ag)-label    -   2. Solid phase-Ab₂ (anti-Ag)+Ag/Ab₁ (anti-Ag)-label complex-   Direct antibody assay (i)    -   1. Ab₁ (analyte=anti-Ag)+Ab₂ (anti-Ab₁)-label    -   2. Solid phase-Ag+Ab₁ (anti-Ag)/Ab₂ (anti-Ab₁)-label complex-   Direct antibody assay (ii)    -   1. Ab₁ (analyte=anti-Ag)+Ab₂ (anti-Ab₁)-label    -   2. Solid-phase-Ab₃ (anti-Ag)/Ag+Ab₁ (anti-Ag)/Ab₂        (anti-Ab₁)-label complex-   Indirect antigen assay    -   1. Ag (analyte)+Ab₁ (anti-Ag)+Ab₂ (anti-Ab₁)-label    -   2. Solid-phase-Ab₃ (anti-Ag)+Ag/Ab₁ (anti-Ag)/Ab₂        (anti-Ab₁)-label complex-   Indirect antibody assay (i)    -   1. Ab₁ (analyte=anti-Ag)+Ab₂ (anti-Ab₁)+Ab₃ (anti-Ab₂)-label    -   2. Solid phase Ag+Ab₁ (anti-Ag)/Ab₂ (anti-Ab₁)/Ab₃        (anti-Ab₂)-label complex-   Indirect antibody assay (ii)    -   1. Ab₁ (analyte=anti-Ag)+Ab₂ (anti-Ab₁)+Ab₃ (anti-Ab₂)-label    -   2. Solid phase Ab₄ (anti-Ag)/Ag+Ab₁ (anti-Ag)/Ab₂ (anti-Ab₁)/Ab₃        (anti-Ab₂)-label complex        *Ag indicates antigen        *Ab indicates antibody

A piezoelectric driven printer may be used to dispense precise amountsof multiple disease ligands such as antigens or antibodies or an analyteas a micro array onto a reaction membrane for use in the apparatus ofthe first aspect of the present invention. The ligands or analytes maybe dispensed in particular patterns, e.g. letters for ease ofrecognition of results. Typically, 100 pl of fluid reagent (1 drop), ormultiples thereof, is dispensed, but this will vary depending on theapplication. The resultant size of the spot on the membrane is about 55microns or more in diameter subject to fluid diffusion on the membrane,but again this will vary depending on the application. It is possible todispense droplets with diameters of 5–10 microns, and hence lowervolumes of fluid reagent (for example, 1–10 pl) can be applied. Usingprecise quantitative printing of micro arrays of antibodies, antigens,or other analytes means that tests using precise quantities of thesereagents can be produced for multi disease diagnosis of a single sample.This array technology can be applied to tests for drugs or other markersacross all diagnostic fields.

Alternatively, an adult/neonatal syringe pump 1235 from ATOM MedicalCorporation, Japan, typically used to administrator small quantities ofintravenous liquids through a catheter to hospital patients can beadapted to apply single or multiple lines of a capture analyte to thefirst membrane eg nitrocellulose.

In one preferred embodiment, ligands for detecting tuberculosis, HIV,hepatitis, syphilis and malaria antibodies may be deposited onto areaction membrane. This would allow the simultaneous diagnosis oftuberculosis, HIV, hepatitis, syphilis and malaria from a single bloodsample without the need for intermediate sample treatment steps.

Utilising the present invention allows the assaying of small volumes ofwhole blood and thus the present invention provides a very rapiddiagnostic assay device that is simple to use and can be used in bothlaboratory and point-of-care field diagnostic locations. For example, afinger prick of blood would be sufficient to perform an assay. Similarlylarge volumes of sample can be used in this device by increasing theamount of absorbent material (second member). For instance, 10 mls ofdilute fluids like urine can be can be assayed to detect low abundancemolecules.

Analytes and/or ligands (e.g. antigens or antibodies) can be printeddown in titrating amounts and/or concentrations. Thus, in an individualscreen, this would provide a means of quantitating analyte-ligand levelswithin the sample solution.

The pre-incubation step may also be carried out with a multi-analytedetector where any number of detection analytes can be attached to agold particle or a similar detectable tag e.g. a fluorescent marker.

BRIEF DESCRIPTION OF THE DRAWINGS

A specific embodiment of the present invention will now be described byway of example only and with reference to the accompanying drawings inwhich:

FIG. 1 is a schematic drawing of an apparatus embodying aspects of thepresent invention in a first configuration;

FIG. 2 is a schematic drawing of the apparatus of FIG. 1 in a secondconfiguration;

FIG. 3 is a perspective view of an assay apparatus or cassette embodyingaspects of the present invention;

FIG. 4 is an exploded view of the components of the cassette shown inFIG. 3;

FIGS. 5 a to 5 d show various stages in the use of the apparatus of FIG.3 in carrying out an assay; and

FIG. 6 is a graph comparing test results from samples spiked with alphaamalyse undergoing no-pre-incubation with samples undergoing a oneminute pre-incubation.

BRIEF DESCRIPTION OF A PREFERRED EMBODIMENT

Capture analytes in the form of ligands such as antigens or antibodies(e.g. TB, HIV-1) are printed onto a protein-capture membrane matrix(e.g. a nitrocellulose membrane) in an appropriately sized array usingpiezoelectric chemical printing technology. A suitable chemical printingsystem for use in the present invention involves the use ofpiezoelectric drop-on-demand ink jet printing technology formicro-dispensing fluids in DNA diagnostics or the Combion Inc. synthesisprocess called “CHEM-JET”. To explore drop on demand fluid dispensingfor DNA diagnostics, an eight fluid printer has been developed as partof the Genosensor Technology Development (GTD) project funded by theInstitute of Standards and Technology (USA). Research to date, isfocused on printing oligonucleotide micro-spots onto solid supports. Inthe CHEM-JET technique, which was developed at the California Instituteof Technology, tiny volumes of reagent bearing liquid are squirted ontospecific spots or addresses of a solid substrate much as an ink-jetprinter squirts ink onto a page. By repeatedly returning to each addresswith one or another of a small set of building blocks, in this case,nucleotides modified for the process, huge two-dimensional libraries ofshort DNA chains (oligonucleotides) can be assembled. Such a deviceincluding an imaging means is described in the applicants co-pendingInternational patent application No PCT/AU98/00265, the entire contentsof which are incorporated herein by reference. In the describedembodiment, antigen is printed onto a reaction membrane in 100 pldroplets, or multiples thereof (eg. 10 nl), with each aliquot being 1 mmapart. However, these volumes and distances can be increased/decreasedaccordingly depending on the chosen antigen titre and array size. Forexample, it is possible to dispense droplets with volumes as low as 1–10pl.

In a particularly preferred embodiment, antigens or antibodies can beprinted down in a matrix of dots or lines or in the shape of letters sothat quantitative multiple analyte analysis of a single sample ispossible.

After the dispensed antigen has dried, non-specific protein-bindingsites on the (nitrocellulose) membrane are blocked using 0.5% (v/v)casein in phosphate buffered saline (PBS)+0.05% (w/v) sodium azide+0.1%(v/v) Tween-20 (PBSA wash buffer). It is however an option to leave themembrane unblocked following the printing of the antigen (or antibody)or other ligand.

In another preferred embodiment syringe pump technology used for theadministration of liquids intravenously to patients can be adapted tolay down single or multiple lines on nitrocellulose membranes.

Turning to the drawings, FIG. 1 shows a flow-through assay device 10,which utilises the nitrocellulose membrane described above. The deviceis in the form of a cassette 12 and an associated removable filter frame14. Inside the cassette there is the membrane (typically nitrocellulose)16 on which capture analytes in the form of ligands are printed, asdescribed above, which is located on top of an absorbent matrix 18. Theabsorbent matrix preferably comprises multiple layers of absorbenttissue or an absorbent pad such as blotting paper, in the specificembodiment twenty-four layers (double ply), which have been found topossess an ideal porosity that permits the most rapid flow-through ofvarious solutions. This rapid flow-through is important as it results inlower backgrounds with higher reaction specificity and higher signalresolution.

As shown in FIG. 1, the top of the cassette defines an opening in itsupper face and a depending generally frusto-conical well whose sidesdepend down as far as the membrane 16, to define a chamber havingsloping sides and a base defined by the membrane 16.

The filter unit frame 14 is spaced above the upper surface of thecassette 12. It also defines a depending conical well in the form of achamber 21 also referred to as a “pre-incubation chamber” having slopingsides and a base 22 formed from a 5 μm Whatman grade 1 membrane or a0.22 μm hydrophilic Durapore membrane filter (Millipore, North Ryde,Australia). However, other types of filter/membrane and pore size wouldbe suitable depending on the application. The function of the membraneis to retain a sample to be assayed in the well or pre-incubationchamber 21 long enough for a “pre-incubation step” to take place. Whenmembrane 22 is lowered to contact the membrane 16, capillary attractiondraws the sample from the chamber 20 through membranes 22 and 16 andinto the tissue 18.

For ease of use, two pins 24 are provided which support the filter frame14 at an appropriate distance above the cassette 12 during thepre-incubation step but which allow the filter frame to be pushed downso that the membranes 22 and 16 are in contact for the second stage ofthe process shown in FIG. 2. The frame 14 is also removable so that themembrane 16 can be viewed to determine the results of the assay.

FIGS. 3 to 5 d illustrate one commercial assay device design embodyingthe aspects of FIGS. 1 and 2.

In those Figures, the components which are equivalent to componentsshown in FIGS. 1 and 2 carry the same reference numerals. The cassette12 comprises an upper moulding 12 a and a lower moulding 12 b. Theporous membrane 22 is defined by the base of a pressed filter paperfrustro cone 22 a held in place by a filter retainer 23. The filter unitframe 14 defines two dimples 14 a on which an operator's thumbs maypress when depressing the filter frame to contact the membranes 22 and16.

FIGS. 5 a to 5 d illustrate the stages of operation of the apparatus.FIG. 5 a illustrates the filter frame separate from the cassette 12.FIG. 5 b illustrates the pre-incubation positioned with the base of thechamber/well 21 spaced from membrane 16. FIGS. 5 c and 5 d illustratethe device after the filter unit has been pressed down to bring themembranes 22 and 16 into contact to allow the sample to flow through tothe blotting paper 18.

If the membrane 22 is replaced with a hydrophobic membrane, it ispossible to operate the device with a pre-incubation step solely in theposition shown in FIGS. 3 and 4 with the membranes 22 and 16 always incontact. The hydrophobic membrane 22 will prevent flow of the sample inthe incubation chamber 21 to the reaction membrane 16. After asufficient period of time has past for detection analyte in the chamber21 to bind to the reagent, a suitable wetting agent is added to thesample in the chamber which allows the sample to flow through thehydrophobic membrane past the reaction membrane 16 and into an absorbentmatrix 20.

EXAMPLE 1

Application of the Pre-filter Chamber

Whatman membrane (paper) or Reemay filters (polyester; 1 cm²) areinserted into the chamber 21 in the filter frame to form a conicalretaining vessel (pre-filter unit).

The sample is pipetted into the plastic pre-filter chamber (50–100 ul)along with a detection analyte in the form of a detecting antibody(50–100 ul) bound to colloidal gold (particle size 20–50 nm). The sampleis pre-incubated with the gold-conjugate (O.D.4) within thepre-incubation chamber for thirty seconds after gentle pippetting toensure adequate mixing. After thirty seconds the chamber is pressed intothe well 20 of the test cassette 12. Upon contact with the membrane 16containing the detection zone, the solution filters through to theabsorbent layer 18 beneath. The pre-filter 14 is discarded when thesolution has filtered through and two drops of PBSA wash buffer are thenadded to the reaction membrane to wash away excess gold-conjugaterevealing the results of the assay on membrane 16.

The use of the pre-incubation of the sample with the detection analyteincreases sensitivity by approximately ten fold. Further, anyparticulate matter is retained in the pre-incubation chamber all ofwhich can be removed to provide a clear signal. The use of thepreincubation chamber with the dual roles of permitting a pre-incubationstep and a pre-filtering step, also allows multi-analyte detection onthe reaction membrane by pre-incubating with a multi-analyte probe, e.g.colloidal gold bound to different detecting analytes. In addition,interfering analytes or substances that could cause false positives ornegatives in the assay can be removed or absorbed out in thepre-incubation step, e.g. human antibodies to mouse antigens can beabsorbed out by anti-HAMA antibodies.

Although the above described example relates to the antigens relating todisease, the immunoassay apparatus could be used, for example, as anallergy test kit, as a test kit for drugs of abuse or for analysingnon-human derived samples e.g. bovine, porcine, veterinary tests, andtests in agriculture such as grain quality evaluation, etc.

The method and apparatus of the present invention is particularly suitedto use with swabs which can be simply placed into the chamber 21,swirled around in liquid containing a detecting antibody (50–100 ul)bound to colloidal gold for 30 seconds before the pre-filter unit isdepressed to contact the membranes 22 and 16 together.

Any combination of ligands and analytes can be applied to the system ofthe present invention. The choice of ligands could be tailored to detectprevalent diseases in a particular country or population. For example,analytes from the following combination of diseases could be used fordiagnosis using this array.

1. TB and HIV

2. Hepatitis-B & C, HIV

3. Chagas, HIV, TB, Syphilis and Hepatitis-B & C

4. Malaria, Dengue, TB, Chagas.

Alternatively antigens representing different varieties of wheat orother agricultural products could be printed on the reaction membraneenabling detection of multiple strains with a single test.

EXAMPLE 2

The assay device can also be used for detecting analytes in body fluidsother than blood such as plasma, sera, urine, saliva and sputum. In thissystem, the sample can be retained in the pre-incubation chamber 22 byuse of a hydrophobic membrane such as Reemay or Hollingsworth and Vose7303 instead of the Whatman grade 1 membrane or a 0.22 μm hydrophilicDurapore membrane filter described above. The sample is mixed with thedetection analyte for the required pre-incubation period. To obtainefficient flow through capillary action to the absorbent layer 18 whenthe pre-incubation chamber 22 is lowered onto the cassette 12, one oftwo procedures can be followed:

1. The membrane 16 containing the capture analyte is pre-wet with atleast one drop of wash buffer containing 0.01 M phosphate, 0.15 M NaCl,0.0% Azide, 0.5% Tween 20 or any wetting agent containing a detergent;

2. The membrane 16 containing the capture analyte is blocked with ahygroscopic solution such as sucrose, trehalose, fructose, oralternatively, glycerol. This changes the characteristics of themembrane 16 from a non-hygroscopic to a hygroscopic membrane allowingthe sample to flow through to the absorbent layer 18 upon contact of themembrane at the base of the pre-incubation chamber 22 with membrane 16.

EXAMPLE 3 (COMPARATIVE EXAMPLE)

Comparison of No Pre-Incubation and 1 Minute Pre-Incubation of a SampleSpiked With Alpha Amylase in the Above Described Format

Procedure

A 6% solution of bovine sera albumin was spiked with 0.1 ng/ml, 0.5ng/ml, 1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 500 ng/ml and 1000 ng/mland applied to the above format according to the following procedure:

-   No-preincubation    -   I. The pre-incubation chamber was pressed down so that the base        of the chamber comes into contact with the first member        containing the capture antibody against alpha amylase.    -   II. Sixty microliters of 0.5% tween in saline was added to the        pre-incubation chamber and allowed to filter through to the        absorbent material beneath the first membrane.    -   III. One hundred microliters of spiked alpha amylase sample was        added to the chamber and allowed to filter through to the        absorbent material beneath the first membrane.    -   IV. Sixty microliters of 0.5% tween in saline was added to the        pre-incubation chamber and allowed to filter through to the        absorbent material beneath the first membrane.    -   V. Sixty microliters of anti-alpha amylase antibody linked to        colloidal gold (particle size 20–50 nm) was added to the        pre-incubation chamber and allowed to filter through to the        absorbent material beneath the first membrane.    -   VI. Sixty microliters of 0.5% tween in saline was added to the        pre-incubation chamber and allowed to filter through to the        absorbent material beneath the first membrane.    -   VII. The pre-incubation chamber was removed and the result on        the reaction membrane scanned with a densitometer. Signal        strength was measured in pixel intensity.-   One minute pre-incubation    -   I. Sixty microliters of 0.5% tween in saline was added to first        membrane and allowed to filter through to the absorbent material        underneath.    -   II. The pre-incubation chamber was suspended over the first        membrane so that there was a space between the chamber and the        membrane.    -   III. One hundred microliters of spiked alpha amylase sample and        60 microliters of anti-alpha amylase antibody linked to        colloidal gold (particle size 20–50nm) were incubated in the        pre-incubation chamber for 1 minute.    -   IV. The chamber was lowered until it came in contact with the        first membrane and the mixture of sample and antibody-gold        conjugate allowed to filter through to the absorbent material.    -   V. Sixty microliters of 0.5% tween in saline was added to the        pre-incubation chamber and allowed to filter through to the        absorbent material.    -   VI. The pre-incubation chamber was removed and the result on the        reaction membrane was scanned with a densitometer. Signal        strength was measured in pixel intensity.

Each data point on the graph is the average of two experiments using theapparatus described above. The results show that pre-incubation of thesample with the detection analyte has a minimal detection limit definedin pixel density of around 500 pg/ml of alpha amylase. This is comparedto a minimum detection limit without the pre-incubation of about 50ng/ml and indicates the pre-cubation increases the sensitive by around10 fold.

EXAMPLE 4 (COMPARATIVE EXAMPLES)

Demonstration of Increased Sensitivity With Increased Pre-Incubation ofthe Sample With the Detection Analyte

Samples of amylase diluted in 0.5% saline to 400 ng/mL were treated withimmunogold conjugate against amylase and aliquotted onto theflow-through format in different protocols as shown below.

-   A. The sample was added to the format (without a filter present) and    allowed to filter through prior to adding conjugate, followed by an    aliquot of conjugate immediately the sample had passed through the    membrane.-   B. The sample was mixed in the correct proportions with gold    conjugate and aliquotted immediately onto the flow-through format.-   C. The sample was mixed as with protocol B but added to the flow    through format after a 60 second interval. The results presented in    pixel intensity are shown in the tables below (for 2 experiments):

Sample Control Sample Control S/PC Protocol peak peak area area ratio A82 286 657 2120 317 B 288 758 2062 5509 383 C 823 949 5843 6765 884 A 89516 588 3890 588 B 482 830 3736 6345 602 C 708 829 4506 5822 792

Clearly there is a significant increase in the sample signal when theanalyte is preincubated with the conjugate probe, as distinct tosequential detection on the flow-through format. The difference indetection levels (for the 400 ng/mL sample) equated to between a7.5-fold to 10-fold increase in detectable amylase in the flow throughformat when the sample is preincubated separately to the detectingcapture antibody.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1. A vertical flow through assay test apparatus comprising: a firstmember comprising a first, porous, membrane to which is bound a captureanalyte for binding to a reagent to be detected, the member having anupper surface and a lower surface; a second member being a body ofabsorbent material disposed below and touching the lower surface of thefirst member; a vessel for containing a liquid sample spaced above thefirst member said vessel having side walls and a base, the base beingdefined by a second membrane, the vessel being capable of retaining aliquid sample for a predetermined incubation period; and means forsupporting the vessel above the first member in two positions, a firstposition in which the membrane is spaced a sufficient distance from thefirst member so as to not permit fluid transfer from the vessel to thebody of absorbent material, and a second position in which the secondmembrane is in contact with the first member, such contact permittingfluid transfer from the vessel through the first and second membranes tothe body of absorbent material.
 2. An apparatus as claimed in claim 1wherein the second membrane is a hydrophilic membrane.
 3. An apparatusas claimed in claim 2 wherein the first membrane is a nitrocellulosemembrane.
 4. An apparatus as claimed in claim 1 wherein the secondmembrane is a hydrophobic membrane.
 5. An apparatus as claimed in claim1 wherein the capture analyte is a ligand.
 6. An apparatus as claimed inclaim 5, wherein the ligand is an antigen or an antibody.
 7. Apparatusas claimed in claim 1 wherein the capture analytes comprise ligandsselected from the group consisting of ligands for detectingtuberculosis, ligands for detecting HIV, ligands for detectinghepatitis, ligands for detecting syphilis and ligands for detectingmalaria.
 8. Apparatus as claimed in claim 7 wherein the first membranecarries capture analytes for detecting more than one disease.
 9. Amethod for assaying for the presence of a pre-determined reagent in aliquid sample comprising the steps of: a) providing a first porousmembrane to which capture analytes for binding to the reagent have beenbound; b) placing a liquid sample to be assayed and a detection analytein a vessel having walls and having a base defined by a second porousmembrane, the vessel being capable of retaining the liquid sample for apredetermined incubation period; c) allowing a sufficient period of timeto pass for the detection analyte to bind to the reagent, if present inthe liquid sample; d) contacting the base of the vessel with the firstporous membrane; e) causing the liquid sample to flow through themembranes to allow the reagent to bind to the capture analyte carried onthe first membrane; and f) removing the vessel and inspecting the firstmembrane for binding of the detection analyte, wherein said bindingindicates the presence of the pre-determined reagent in the liquidsample.
 10. A method as claimed in claim 9 further comprising: washingthe first membrane with a buffer prior to inspecting the first membranefor the presence of the detection analyte.
 11. A method as claimed inclaim 9 further comprising the steps of: washing the first membrane witha buffer by addition to the vessel before removal and inspection of thefirst membrane for the presence of the detection analyte.
 12. A methodas claimed in claim 9 wherein the sample is whole blood wherein wholered blood cells are removed in the vessel by the second membrane actingas a filter and plasma in the blood is allowed to flow-through to thefirst membrane.
 13. A method as claimed in claim 9 wherein the samplecontains particulate materials selected from the group including grainextracts, cell or microbial extracts wherein particulate materials areremoved in the vessel by the second membrane acting as a filter.
 14. Amethod as claimed in claim 9 wherein the sample comprises body fluidssuch as plasma, sera, urine, saliva and sputum and wherein the secondmembrane is a hydrophobic membrane.
 15. A method as claimed in claim 9including the step of removing or neutralising unwanted analytes in thesample that may interfere with the binding of the reagent with thecapture analyte on the first membrane in the vessel.
 16. A method asclaimed in claim 15 wherein antibodies or other analytes which bind tothe unwanted analytes are bound to the walls or the base of the vessel.17. A method for assaying for the presence of a pre-determined reagentusing a vertical flow through assay test apparatus comprising: a firstmember comprising a first, porous, membrane to which is bound a captureanalyte for binding to a reagent to be detected, the member having anupper surface and a lower surface; a second member being a body ofabsorbent material disposed below and touching the lower surface of thefirst member; a vessel for containing a liquid sample spaced above thefirst member said vessel having side walls and a base, the base beingdefined by a second membrane, the vessel being capable of retaining aliquid sample for a predetermined incubation period; and means forsupporting the vessel above the first member in two positions, a firstposition in which the membrane is spaced a sufficient distance from thefirst member so as to not permit fluid transfer from the vessel to thebody of absorbent material, and a second position in which the secondmembrane is in contact with the first member, such contact permittingfluid transfer from the vessel through the first and second membranes tothe body of absorbent material, comprising the steps of: a) placing asample to be assayed and a detection analyte in the vessel, with thevessel disposed in the first position; b) allowing a sufficient periodof time to pass for the detection analyte to bind to the reagent, ifpresent; c) depressing the vessel to the second position to contact thebase of the vessel with the first porous membrane; d) allowing thesample to flow through the first and second membranes to allow thereagent, if present to bind to the capture analyte carried on the firstmembrane; and e) removing the vessel and inspecting the first membranefor binding of the detection analyte, wherein said binding indicates thepresence of the pre-determined reagent in the liquid sample.
 18. Amethod as claimed in claim 17 wherein the second membrane is hydrophobicand the step of allowing the sample to flow through the first and secondmembranes includes the addition of a wetting agent on the upper surfaceof the second membrane prior to moving the vessel to the secondposition.
 19. A method for assaying for the presence of a pre-determinedreagent using a vertical flow through assay test apparatus comprising: afirst member comprising a first, porous, membrane to which is bound acapture analyte for binding to a reagent to be detected, the memberhaving an upper surface and a lower surface; a second member being abody of absorbent material disposed below and touching the lower surfaceof the first member; a vessel for containing a liquid sample spacedabove the first member said vessel having side walls and a base, thebase being defined by a second membrane which is a hydrophobic membrane,the vessel being capable of retaining the liquid sample for apredetermined incubation period; and means for supporting the vesselabove the first member in two positions, a first position in which themembrane is spaced a sufficient distance from the first member so as tonot permit fluid transfer from the vessel to the body of absorbentmaterial, and a second position in which the second membrane is incontact with the first member, such contact permitting fluid transferfrom the vessel through the first and second membranes to the body ofabsorbent material, said method comprising the steps of: a) placing asample to be assayed and a detection analyte in the vessel; b) allowinga sufficient period of time to pass for the detection analyte to bind tothe reagent, if present; c) adding a wetting agent to the vessel; d)allowing the sample to flow through the first and second porousmembranes to allow the reagent, if present, to bind to the captureanalyte carried on the first membrane; and e) removing the vessel andinspecting the first membrane for binding of the detection analyte,wherein said binding indicates the presence of the pre-determinedreagent in the liquid sample.